Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
(N/A)
Non Technical Summary
Trimethylamine N-oxide (TMAO) is a harmful metabolite derived from gut bacteria. Circulating levels of TMAO are positively associated with atherosclerosis risk. Cardiovascular disease, including atherosclerosis, is the leading cause of death worldwide. No drugs are approved to lower TMAO. Strategies such as diet represent our best bet for controlling TMAO. Dietary phenolics have shown potential for beneficially altering gut bacterial communities and metabolism. One such compound is chlorogenic acid, found in foods such as blueberries. Our preliminary data suggest that chlorogenic acid-rich blueberries reduce TMO levels in vivo. However, chlorogenic acid content varies widely in blueberry cultivars. We hypothesize that the chlorogenic acid content of blueberries corresponds to their TMAO-lowering and cardioprotective effects. We propose to use in vitro and in vivo studies to determine the relationship between the chlorogenic acid content of blueberries and their TMAO-lowering and anti-atherosclerosis activities. The rationale for this work is that understanding whether the chlorogenic acid content of foods correspond to their efficacy for lowering TMAO can lead to healthier dietary patterns in at-risk individuals and the population, and that breeders can use these data to breed chlorogenic acid-rich cultivars. The long-term effects of such patterns may reduce cardiovascular disease burden and increase quality of life at the individual and population levels. This application responds to Program Area Priority 3d (Food and Human Health) by investigating the impact of food components (blueberry chlorogenic acid) on the gut microbiome and its metabolites (bacterial-derived TMAO) and how this relates to human health (prevention of atherosclerosis).
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Cardiovascular disease (CVD) causes ~32% of deaths worldwide. There is an urgent need to develop complementary strategies across diet, lifestyle, and medicine to reduce CVD incidence and burden. Elevated blood trimethylamine N-oxide (TMAO) concentrations increase atherosclerosis risk. TMAO is formed through a microbial-host axis involving release of trimethylamine (TMA) from choline by specific gut bacteria containing the cutC/D gene cluster, which encodes choline TMA lyase. TMA is absorbed into the circulation and then oxidized to TMAO by hepatic flavin-containing monooxygenase 3 (FMO3). TMAO is thought to promote the development of atherosclerosis. There is no FDA-approved drug to lower TMAO levels, underscoring the need for complementary strategies to manage TMAO production. Interest is emerging in the TMAO-lowering activities of dietary phenolics. Phenolics may reduce TMAO levels by reducing the abundance of bacterial genera that convert choline to TMA, or direct inhibition of TMA lyase. Through our ex vivo-in vitro anaerobic fecal fermentation and in vivo mouse studies, we identified chlorogenic acid (CGA) as a TMA production inhibitor. Blueberries are rich in CGA and have known cardioprotective benefits. CGA has been evaluated as a TMA/TMAO production inhibitor in a few pre-clinical studies. Our preliminary data indicate that CGA inhibits choline conversion to TMA in a dose-dependent manner. Importantly, levels of CGA vary widely across blueberry cultivars, but it is unknown whether variation in CGA level contributes to different TMAO-lowering and cardioprotective activities from blueberries. Further data are required to confirm the potential of CGA-rich blueberries as TMA/TMAO production inhibitors in vivo and to determine if more CGA content imparts greater activity. To advance our ability to employ CGA-rich blueberries to lower circulating TMAO and reduce atherosclerosis risk, it is critical that we determine whether CGA levels in blueberries determine their efficacy and elucidate the mechanism(s) by which this occursOur overall objective is to evaluate whether CGA is a key bioactive that enables blueberries to inhibit formation of the pro-atherogenic gut microbial metabolite TMAO (and its precursor, TMA) when consumed. Our central hypothesis is that CGA content is a primary determinant of the TMAO-lowering benefits of blueberries. Our specific objectives are to:Determine if blueberry CGA content predicts reduced TMAO formation and atherosclerosis when cultivars with >10-fold difference in CGA are administered to rodents;Determine whether CGA content correlates with reduced TMA production in multiple blueberry accessions from a genetic diversity population with a large spread of CGA in our ex vivo-in vitro human fecal fermentation model; andElucidate mechanism(s) by which CGA and CGA-rich blueberries modulate TMA formation in the gut using isolated bacteria known to carry cutC/D.
Project Methods
Blueberry material. Access to blueberry germplasm with wide variation in chlorogenic acid (CGA) is critical for this project. The National Clonal Germplasm Repository (NCGR) maintains a large collection of blueberry germplasm. We will obtain material from the 2023 harvest year fruit from the diversity panel. Blueberry CGA, anthocyanins and other phenolics such as flavan-3-ols will be quantified by LC-MS/MS. We will also measure total phenolics (Folin), flavan-3-ols (DMAC), and monomeric anthocyanins (pH-shift).Objective 1. Determine if blueberry CGA predicts inhibition of TMAO formation and atherosclerosis when cultivars with >10-fold difference in CGA are administered to rodents.Hypothesis and Approach: We will use rodent models to test the impact of blueberry cultivars with different CGA content on TMA/TMAO production acutely and chronically (Exp. 1-A-B rats) and on atherosclerosis development (Exp. 2, ApoE−/− mice). We hypothesize that 1) blueberry supplementation will reduce TMAO and prevent atherosclerosis, and 2) CGA-rich blueberries will be more effective than CGA-poor blueberries.Experiment 1. Compare efficacy of CGA-rich and CGA-poor blueberries for inhibiting TMA and TMAO production in vivo. We will perform both acute and chronic experiments, enabling us to probe distinct mechanisms: direct TMA lyase inhibition in acute experiments (Exp.1A) vs. altered microbiome composition, cutC/D copy numbers and FMO3 expression (not inhibition) in chronic experiments (Exp. 1B). Identifying mechanism(s) of action is critical for the subsequent design of optimal dietary strategies to maximize the effects of blueberry consumption.Exp. 1A: Acute Effects. We will first compare the acute efficacy of CGA-rich vs. CGA-poor blueberries to reduce TMA and TMAO. We will employ a randomized crossover design to compare the acute efficacy of CGA-rich or CGA-poor blueberry powders and pure CGA matched to CGA content of blueberry powders. We hypothesize that blueberries and CGA will lower TMA and TMAO in vivo, and that higher doses of CGA) will result in greater inhibition. Exp. 1B: Chronic Effects. Chronic CGA supplementation improves the gut microbiome and protects against CVD in animal models. CGA-rich blueberries may reduce TMA and TMAO by mechanisms requiring chronic exposure, including shifts in microbiome taxonomic distribution, altered levels of cutC/D, and/or changes to FMO3 expression. We will perform a chronic supplementation study to assess these mechanisms. We hypothesize that chronic consumption of blueberries and CGA will reduce TMA and TMAO in a dose-dependent manner.Experiment 2. Determine whether the CGA content of blueberries affects their ability to prevent or delay atherosclerosis in choline-fed ApoE-/- mice. We hypothesize that blueberries will prevent development of atherosclerosis in choline-fed ApoE-/- mice concurrent with suppressing TMAO, and that CGA-rich blueberries will be more effective than CGA-poor blueberries at both. We will determine whether blueberries inhibit atherosclerosis and reduce TMAO levels in an established atherosclerosis model (ApoE-/- mice) fed 1% choline.Objective 2. Determine whether blueberry CGA content correlates with inhibition of choline conversion to TMA in multiple accessions from a genetic diversity population with a large spread of CGA in our validated ex vivo-in vitro human fecal fermentation modelHypothesis and Approach: We will perform fecal fermentations to build on our preliminary in vitro data that CGA-rich blueberries inhibit the microbial generation of TMA201,215,239 (Fig. 10), and establish whether blueberry CGA content exerts a dose-dependent effect. We hypothesize that CGA content will strongly correlate with reduced TMA production by gut bacteria.Experiment 3. Screen blueberry cultivars with large variation in CGA content for inhibition of choline-d9 conversion to TMA-d9 by human fecal bacteria. We will perform fermentations per our methods to assess the inhibition of choline-d9 conversion to TMA-d9 by human fecal bacteria. Feces from healthy donors will be obtained from OpenBiome (Cambridge, MA).Exp. 3A: Blueberry Fermentations. To identify the role of CGA for reducing TMA production, we will study 15-20 blueberry cultivars from the genetic diversity panel discussed above with a wide range of CGA levels. We will subject blueberries to simulated oral, gastric and small intestinal in vitro digestion by our established protocols (equiv. to 0.5-1 servings/2 L upper GI volume) and then ferment them with choline-d9 substrate to compare efficacy for inhibiting TMA-d9 production vs. blank digesta and each other. We hypothesize that blueberry CGA levels correlate to inhibition of TMA production. We will perform linear regression analyses using CGA content (independent variable) and TMA inhibition outcomes such final TMA levels, AUCs, % TMA inhibition vs control, etc. (dependent variables).Exp. 3B: Berry Component Fermentations. We will isolate the effects of CGA and other phenolics vs. non-phenolic blueberry components by preparing CGA/phenolic-rich extracts from blueberries and fermenting these extracts vs. the unextracted material (polysaccharides, proteins, lipids, etc.). We will digest and ferment the phenolic extract and unextracted material separately and compare them to each other and whole digesta to identify effects of phenolics vs. other material (fiber, etc.). Similar analyses will be performed to those for Exp. 3A.Exp. 3C: Inter-Individual Variability. To increase translational relevance, we will assess the TMA inhibition capacity of pure CGA and the berry powders highest and lowest in CGA across human fecal donors. This will suggest translational feasibility to diverse human populations and assist in powering human trials.Objective 3: Elucidate mechanism(s) by which CGA and CGA-rich blueberries modulate TMA formation in the gut using isolated bacteria known to carry cutC/DHypothesis and Approach: We will interrogate bacterial mechanisms underlying the TMA-lowering effects of CGA-rich blueberries by performing fermentations with bacterial genera known to carry the cutC/D gene cluster. We hypothesize that CGA-rich blueberries directly inhibit TMA lyase or reduce cutC copy numbers or viability of bacterial species carrying cutC. We also hypothesize that CGA content of berries is directly correlated to activity.Experiment 4. Evaluate mechanisms by which CGA-rich blueberries inhibit bacterial TMA production. We will assess the potential for blueberries, pure CGA, phenolic extracts, etc. to directly inhibit choline TMA lyase. Direct inhibition requires that the inhibitory molecule(s), substrate, and enzyme be simultaneously present. Exp. 4A. Isolated live bacterial fermentations. We will assess the potential for blueberry cultivars to inhibit TMA lyase. We will perform fermentations with gut bacterial strains that carry cutC/D. We hypothesize that inhibitory effects of CGA and blueberries in fecal slurries will be reproduced with isolated bacterial strains, and that TMA lyase inhibition and/or or growth inhibition of TMA producers are mechanisms of action of CGA-rich blueberries. Exp. 4B. TMA lyase inhibitory experiments in lysed (non-viable) bacteria. A limitation to assessing enzyme activity in live cells is that alterations to growth, metabolic activity, viability, or community composition may confound direct enzyme inhibition. To overcome this, we will assay TMA lyase inhibition in non-viable cell lysates from isolated bacteria used above. We will prepare lysates to obtain cell-free TMA lyase, screen lysates to confirm TMA lyase activity, and then perform inhibitory assays with blueberry cultivar or CGA digestas as described above. Effects in lysed (non-viable) cells are due to TMA lyase inhibition and not altered bacterial viability, growth, cutC/D numbers or expression